Patients and samples
This study was approved by the Institutional Review Board of the Fudan University Shanghai Cancer Center, and informed consent was obtained from all patients. The expression of BAI1 in TNBC patients was detected using immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting (WB). A tissue microarray (TMA) with 120 paraffin-embedded TNBC samples were performed to identify the expression levels of BAI1 in TNBC patients. qRT-PCR was performed to examine BAI1 mRNA expression in 159 breast cancer with different subtypes, 63 paired TNBC tumor samples and adjacent normal tissues (ANTs). Western blotting was performed to examine BAI1 protein expression in eight paired TNBC tumor samples and ANTs.
Analysis of Publicly Available Datasets
To analyze mRNA expression of BAI1 in whole breast cancer samples and different subtypes, and the effect of BAI1 expression on the prognosis of patients with breast cancer, the Gene Expression Profiling Interactive Analysis (GEPIA) online tool (http://gepia.cancer-pku.cn) were used to analyze mRNA expression of BAI1 and prognostic value in BC samples from TCGA provisional dataset.
Cell culture and reagents
Human triple negative breast cancer cell lines (MDA-MB- 231, MDA- MB- 453, MDA- MB- 468, Hs578T, BT549 and BT20), and human embryonic kidney 293T (HEK293T) cell line were kindly provided by Dr. Daqiang Li and were maintained according to standard American Type Culture Collection (ATCC) protocols. All cell lines were cultured in high- glucose DMEM medium containing 10% FBS and 1% penicillin/streptomycin in a 37°C humidified incubator with 5% CO2.
Cisplatin (purchased from MCE) was suspended in dimethyl sulfoxide (DMSO). Cells were seeded in 96-well plates with 100 μl of medium at a concentration of 4000 cells/well. DMSO was used as the negative control. The cells were then cultured for 48h and then tested using a CCK-8 kit to measure the half maximal inhibitory concentration (IC50).
The antibody for BAI1 (ab135907) and antibodies targeting GAPDH (ab181602) were purchased from Abcam. Antibodies targeting MDM2 (Cell Signaling Technology, CST#86934), p73 (CST#14620), p21 (CST#8831), Cyclin A2 (CST#4656), Cyclin B1 (CST#4138), BCL2 (CST#15071), BAX (CST#5023), Flag (CST#14793) and IgG (CST#5415) were purchased from CST. The antibodies used in this article are listed in (Additional file 1: Table S1).
Slides were dewaxed at 60 °C for 2 h followed by three washes with xylene and rehydrated with different concentration gradients of ethanol, then washed with distilled water for 10 min. For antigen retrieval, slides were put into the ethylene diamine tetraacetic acid buffer (EDTA, pH 6.0) for 4 min when the buffer was heated to 100 °C. Then inhibited the endogenous peroxidase activity by 3% hydrogen peroxide for 30 min. After that, the slides were incubated with anti-BAI1 antibody (1:100 dilution; Abcam, Shanghai, China) overnight at 4 °C. The slices were incubated with a biotin-labeled secondary antibody for 30 min, then followed by 3, 3′-diaminobenzidine (DAB; Zhongshan biotech) substrate.
For each sample, IHC staining was scored by two pathologists using the immunoreactive score (IRS), which is based on the staining intensity and the percentage of positive cells. The staining intensity was scored 0–3 (0 = negative; 1 = weak; 2 = moderate; 3 = strong), the percentage of positive stained cells was also scored into four categories: 1 (0–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%), then staining pattern was defined as negative (IRS: 0), weak (IRS: 1–3), moderate (IRS: 4–6), and strong (IRS: 8–12).
RNA extraction and quantitative real-time PCR
Cells were collected, and total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Reverse transcription (RT) was conducted using PrimeScript RT Master Mix (Takara, Dalian, China) to synthesize cDNA. qRT–PCR was performed with SYBR Green PCR Master Mix (Takara, Dalian, China) on an ABI 7900HT (PE Applied Biosystems) qPCR machine. For relative quantification, target gene mRNA expression was normalized to GAPDH expression. The primers used in the qRT-PCR assays are listed in (Additional file 1: Table S2).
Cells were collected and lysed with RIPA buffer (Thermo Scientific) supplement with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Cell lysates was centrifugated at 12,000 g for 15 min at 4 °C, the protein supernatants were collected and quantified using a bicinchoninic acid assay (Thermo Scientific) according to the manufacturers’ instruction, then proteins were subjected to SDS-PAGE, and transferred onto PVDF membranes (Millipore, Billerica, USA). after incubated with blocking buffer (5% non-fat dry milk in TBST) for 1 h, PVDF membranes were cultured in indicated primary antibodies overnight at 4℃. After three washes with TBST and incubated with HRP-conjugated secondary antibodies for 1 h, antibody detection was conducted using an enhanced chemiluminescent substrate kit (Yeasen).
RNA interference, plasmid transfection, and lentivirus transduction
BAI1 siRNA sequences and negative controls were designed by GenePharma (Shanghai, China) and used to transfect cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The target sequences of BAI1 siRNAs are listed in (Additional file 1: Table S3). For overexpression, full-length BAI1 and p73 were identified and cloned to generate Flag-BAI1 and His-p73 constructs. To construct stable BAI1 overexpression cells, lentivirus was constructed and used to infect MDA-MB-231 and MDAMB-468 cells, which were then selected with puromycin.
Cell viability and colony-formation assay
Cells were seeded in 96-well plates and cell viability was assessed using a Cell Counting Kit-8 (CCK8; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. For colony-formation assays, 1000 viable cells were seeded in 6-well plates and cultured for 2 weeks. Colonies were fixed with ethanol and stained with 1% crystal violet before being counted.
Flow cytometry analysis
Cell cycle and cell apoptosis analyses were performed via flow cytometry on a FACS can instrument (Beckman Coulter, Brea, CA, USA). For the cell cycle analysis, cells were harvested, and fixed with 70% ethanol at 4 °C overnight, and stained with propidium iodide (50 μg/ml), containing 100 μg/ml RNase A for 15 min. For apoptosis analysis, cells were harvested, washed with PBS, and incubated with Annexin V-PE and 7-AAD (BD Biosciences, San Diego, CA, USA).
Nuclear cytoplasmic fractionation
The extraction and isolation of nuclear and cytoplasmic protein were performed by using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions.
Cells were washed 3 times in PBS and then fixed with 4% paraformaldehyde for 15 min, permeabilized in 0.3% Triton X-100 for 15 min and blocked with 10% goat serum for 1 h at room temperature. Then, cells were incubated with primary antibodies overnight at 4 °C, washed 3 times in PBST, and incubated with appropriate secondary antibody conjugated with Alexa 555 (red) or Alexa 488 (green) (Cell Signaling Technology), respectively. DAPI (Abcam, Shanghai, China) was used to stain nuclei and mount cells. A Leica SP5 confocal Laser Scanning Microscope (Leica Microsystems, Buffalo Grove, USA) was used to capture and analyze images.
MDA-MB-231 and MDA-MB-468 cells infected with Flag-BAI1 were lysed on ice in NP-40 lysis buffer (50 mM Tris-HCl, pH 8, 150mM NaCl, 0.5% NP- 40, 10% glycerol, 2mM MgCl2, and 1mM EDTA) supplemented with containing 1× protease inhibitor cocktail (Selleck, B14001) and 1× phosphatase inhibitor cocktail (Selleck, B15001). Cell lysates were incubated with 3 μg anti-BAI1 or IgG antibody (Cell Signaling Technology) overnight at 4 °C, then added 50μl protein A/G Sepharose beads (CST#70024) in the cell lysates and incubated for 4h at 4 °C. Immuno-complexes were washed with lysis buffer three times before being resolved by SDS-PAGE and detected by western blot using the indicated antibodies.
Luciferase reporter assays
Cells were co-transfected with 1μg of the indicated firefly luciferase reporter plasmid (p21 or BAX) plus the renilla luciferase control vector, with or without 1μg of the indicated expression plasmid (p73) in the presence or absence of BAI1. Cell lysates were prepared and quantified using Firefly & Renilla Dual luciferase assay (Yeasen) after transfection (48h later) according to the manufacturer's recommendations.
Statistical analyses were performed using IBM SPSS 20.1 and GraphPad Prism v 8.0 (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation from at least three independent experiments. Two-tailed Student t tests or a one-way ANOVA was applied to assess difference between or among different groups. The chi-square test was used to analyze the relationship between BAI1 protein expression and clinicopathologic parameters. The Kaplan– Meier method and log-rank test were used for survival analysis. Spearman rank correlation test was used to calculate correlation coefficients, and p< 0.05 was considered to indicate statistical significance.